PROJECT SUMMARY Osteoporosis is one of the major contributors to the overall physical and psychological manifestations of frailty and suffering associated with aging. Adult bone mass is determined by the balance between bone formation by osteoblasts (OBs) and bone resorption by osteoclasts (OCs). A perturbation of this equilibrium leads to osteoporosis. The majority of existing therapeutics for osteoporosis is limited to the inhibition of OCs, thus the development of new agents that are anabolic for bone in the setting of osteoporosis is critical. Additionally, current anabolic agents, such as intermittent parathyroid hormone (PTH) and anti-sclerositn antibody, are both limited in use due to the fear of PTH-induced bone tumors and an increase in TNF-dependent inflammation, respectively. We have discovered that the adaptor protein Schnurri-3 (SHN3) profoundly suppresses new bone formation. Mice lacking SHN3 develop a progressive increase in bone mass due to the augmented activity of OBs through enhanced Wnt signaling. Importantly, SHN3 inhibition prevents bone loss in an osteoporosis by estrogen deficiency mouse model and is not associated with phenotypes in non-skeletal tissues. Finally, mesenchymal stem cells (MSCs) are a promising mechanism for the repair of bone loss in osteoporosis. These cells may be genetically and metabolically manipulated by deleting SHN3 and labeling them with bone- targeting probes to augment their repair systemic bone loss potential in this setting. In this R21 application, using our new cell metabolic labeling strategy, we will test the hypothesis that SHN3 is a novel target to promote bone formation in osteoporosis and will identify molecules to regulate the SHN3 pathway in this setting. Aim 1 will optimize the bone-homing capability of SHN3-deficient MSCs. To improve a selective delivery of MSCs to bone in vivo, the development will focus on the most compatible conditions for MSC functionalization, the optimal bone homing composition, and tracking reporters in vivo. The engineered MSCs under these optimized conditions will be introduced into mice via systemic infusion and examined for bone homing and retention in vivo. Aim 2 will test whether bone-seeking SHN3-deficient MSCs can promote the healing of bone loss in osteoporosis. We will determine in Part A whether the transplanted, bone-homing SHN3-deficient MSCs can restore bone loss in mouse models of osteoporosis, ovariectomized mice (estrogen deficiency-induced bone loss) or aged mice (aging-induced bone loss). Part B will identify potential novel regulators promoting bone formation in osteoporosis in the absence of SHN3. Osteogenic and/or osteoinductive factors produced by transplanted, SHN3-deficient MSC-lineage OBs (cy7-label) and responsive genes to these factors in mature OBs in host mice (GFP-expression) will be identified by transcriptome analysis. The targets identified by these multiple approaches will be prioritized for validation. This data will provide proof-of-principle that transplanted, bone-homing MSCs with SHN3 deficiency can restore bone loss in osteoporosis, and identify new targets to promote anabolic bone formation in this setting.